It has been known for some time that some of the hormone peptides can be derived from the glands of land and aquatic animals and that such hormones can be used to treat hormonal deficiencies in man and in animals. The adrenocorticotropic hormone is effective in treating deficiencies of this hormone, and calcitonin is effective in the treatment of Paget's Disease of the bone (see Seminars in Drug Treatment, Vol. 2, No. 1, 1972).
More recently, methods for the synthesis of substance containing hormonal activity have been discovered. For example, see Colescott et al, U.S. Pat. No. 3,915,949 disclosing the synthesis of adrencorticotropic hormone and U.S. Pat. No. 3,926,938 relating to the synthesis of calcitonin.
In order to utilize the crude materials which come from the synthesis of peptide hormones in the treatment of humans, it is necessary to purify the materials which come as a result of the synthesis. The classical methods heretofore used for this purification are tedious and time consuming, and usually result in low yields of the hormones. These classical methods are adaptable only to very small quantities of the materials. Examples of the methods heretofore used are found in Pat. No. 3,915,949 in Column 18 and Pat. No. 3,926,938 in Column 20.
We have sought new and improved methods for conducting the necessary purification of crude peptide hormone materials. We are aware that partition chromatography has been suggested for purifying certain hormone peptides (see Journal of Chromatography, pages 423-428; Elsevier Publishing Company, Amsterdam, Holland; CHROM. 5708).
In general, the procedure involved in carrying out partition chromatography is well known. This procedure includes packing a column, suitably using a fine dextran resin such as is sold under the trademark SEPHADEX. The resin may be prepared for use in the column by adding 4.5 kg. of the resin to 35 liters of 0.2 M acetic acid in a container and bringing the volume up to 40 liters to make an 8.5:1 ratio of solvent to resin. Some of the supernatant may be removed, resulting in about 5 liters of supernatant and 23 liters of suspended resin.
To prepare the column, the prepared resin may be poured into the column as a thick slurry. The volume of the resin at this stage (in liters) may be about five times its weight (in kilograms). The resin may be allowed to settle under a flow of 0.2 M acetic acid until it reaches a constant height.
The procedure of partition chromatography involves three essential steps in the following sequence:
(1) The equilibration of the column to lower phase, PA1 (2) The equilibration of the column to upper phase, and PA1 (3) The separation of the mixture into its components based on the individual differential partition coefficients of the components between the two phases of an immissible mixture of solvents. PA1 A Folin-Lowry analysis of the collected fractions demonstrated that no separation of impurities from the desired hormone human calcitonin was achieved. The impurities and the human calcitonin co-eluted, and no separation was observed.
The term "partition chromatography" when used herein, is taken to mean a purification procedure involving these three steps.
In an attempt to purify crude adrenocorticotropic hormone (ACTH) we obtained a quantity of this crude hormone prepared in accordance with the Colescott et al. U.S. Pat. No. 3,915,949 and subjected the crude hormone to a purification procedure by partition chromatography. This attempt and the results obtained are reported in the following paragraph:
A solution of 200 mg. of the crude ACTH prepared in accordance with the process of Pat. No. 3,915,949, in 20 ml. of upper phase from the system 1,200 ml. of n-butanol, 300 ml. of ethanol and 1,500 ml. of 0.2 M ammonium acetate containing 0.6 ml. of acetic acid, was placed on a dextran resin column, previously equilibrated with lower and then to upper phases of the solvent system, and was then eluted with upper phase. The effluent was collected in tubes on a fraction collector. A Folin-Lowry analysis showed that only very small traces of peptide had been eluted. This indicated to us that this partition system does not effectively move the ACTH down the column in the partition chromatography procedure. No improvement in the purity of the crude ACTH could be detected by thin layer chromatography.
We have also tried to purify crude human calcitonin using partition chromatography with substantially the same solvents and procedures as we have described in our attempts to purify ACTH. A description of these attempts is reported as follows:
From the above, it is apparent that our attempts to purify crude human calcitonin using this partition chromatography solvent system did not produce a satisfactory purification process.